Numerous fields of application require determining a multiplicity of biologically relevant analytes in a complex sample, for example in diagnostic processes for determining the state of health of an individual or the effect of a therapeutic treatment or in pharmaceutical research and development for determining the influence of biological systems such as, for example, an organism and the complex mode of action thereof by external actions such as, for example, by means of supplying biologically active compounds.
While known analytical separation methods are usually optimized in order to fractionate a very large number of compounds present in a given sample according to a predefined physicochemical parameter such as, for example, the molecular weight or the quotient of molecular charge and mass, in as short a time as possible, bioaffinity assays are based on using in each case one biological or biochemical or synthetic recognition element of very high specificity, in order to recognize and bind the corresponding (individual) analyte in a sample of complex composition in a highly selective manner. Detection of a multiplicity of different compounds thus requires the use of a corresponding number of different specific recognition elements.
An assay based on bioaffinity reactions may be carried out both in a homogeneous solution and on the surface of a solid support. Depending on the specific design of the process, the latter requires, after binding of the analytes to the corresponding recognition elements and, where appropriate, further detection substances and also, where appropriate, between various process steps, in each case washing steps in order to separate the produced complexes of said recognition elements and the analytes to be detected and also, where appropriate, further detection substances from the rest of the sample and the optionally employed additional reagents.
Processes for simultaneously detecting a multiplicity of different nucleic acids in a sample with the aid of corresponding complementary nucleic acids immobilized on a solid support in discrete, spatially separated measurement areas as recognition elements are now relatively widespread. For example, arrays of oligonucleotides as recognition elements, which are based on simple glass or microscope slides and which have a very high feature density (density of measurement areas on a shared solid support), have been disclosed. U.S. Pat. No. 5,445,934 (Affymax Technologies), for example, describes and claims arrays of oligonucleotides having a density of more than 1000 features per square centimeter.
Recently, descriptions of arrays and assays of a similar kind carried out therewith for simultaneously determining a multiplicity of proteins, for example in U.S. Pat. No. 6,365,418 B1, in particular using arrays of immobilized antibodies as recognition elements for the analytes to be detected, have become more frequent.
The patent documents regarding such “microarrays”, for detecting both nucleic acids and other biopolymers such as, for example, proteins, describe in each case that a multiplicity of different specific recognition elements is immobilized in discrete measurement areas to generate an array for analyte recognition, and the sample to be studied containing the analytes (where appropriate in a complex mixture) is then contacted with this “capture array”. According to the descriptions disclosed, different specific recognition elements are present here in each case in a form of the highest possible purity in different discrete measurement areas, and as a result usually different analytes of the sample bind to measurement areas with different recognition elements.
This type of known assay requires purifying and concentrating said specific recognition elements, to be immobilized in a form of the highest possible purity, by means of in parts very complicated steps. Since different recognition elements differ more or less greatly in their physicochemical properties (e.g. in their polarity), there are also corresponding differences in the conditions for optimal immobilization of said recognition elements, for example by adsorption or covalent binding, in discrete measurement areas on a shared solid support, where appropriate on an adhesion-promoting layer applied thereto. Consequently, the immobilization conditions chosen for immobilizing a multiplicity of different recognition elements (such as, for example, type of adhesion-promoting layer) can hardly be optimal for all recognition elements at the same time, but merely be a compromise between the immobilization properties of the various recognition elements. Another disadvantage is the fact that in each case only one supplied sample per array can be studied for analytes present therein in this kind of capture array.
There existed therefore the need for a modified assay design which enables a multiplicity of samples to be studied for analytes present in said samples, either simultaneously in one array or a plurality of arrays on a shared support or sequentially in a plurality of arrays on a plurality of supports. For this purpose it would be convenient to apply the samples to be studied themselves, rather than the different specific recognition elements, in discrete measurement areas in one or more arrays on one or more supports, either directly, i.e. in untreated form, or after as few preparation steps as possible. Such an assay design will be referred to hereinbelow as an “inverted assay architecture”.
To satisfy this need, Paweletz et al. in Oncogene 2001, Vol. 20, 1981-1989, for example, have recently proposed such arrays for protein detection based on an inverted assay architecture under the name “reverse phase protein microarrays”.
A common problem of such processes for analyte detection with the aid of microarrays as well as, in an even more general form, of assays which are carried out on surfaces and which are based on binding reagents binding specifically as specific binding partners to the analytes to be detected is the occurrence of unspecific binding events which are not based on the specific interaction between the analytes and the binding reagents and, where appropriate, further detection reagents used for their detection.
U.S. Pat. No. 5,726,064 describes various methods of compensating interferences of the assay signals by background signals such as, for example, background fluorescence, which may be caused in particular by unspecific binding events, and changes in temperature or pH, which could impair the assay signals observed. These methods are essentially based on providing additional areas designated to such compensation purposes, aside from the areas designated for generating the assay signals, on a shared solid support.
The US patent application 2004/0043508 A1 describes the extent of specific and unspecific binding to different surfaces for preparing capture arrays, which have been treated with different materials to minimize unspecific binding. In this context, coatings with electrostatic action are said to be advantageous for reducing unspecific binding.
U.S. Pat. No. 5,677,196 also describes different surface coatings for minimizing unspecific binding, in particular those comprising polyethylene glycols, and measurements of the absolute and relative proportions of unspecific binding, but again in a (sensor) format corresponding to capture arrays.
Said methods for minimizing the effects of unspecific binding on the assay results share the fact that they are based on altering the nature of the surface of the support, in order to prevent or minimize thereby binding of analytes or other binding and detection reagents used in the assay outside the measurement areas with analyte-specific recognition elements immobilized there. At the same time, it is tacitly assumed that unspecific binding does not occur in said measurement areas themselves; for such effects could not be taken into account with the aid of the processes described. In the case of capture arrays, with a well-defined composition of the compounds applied to the measurement areas, namely usually a standard form of recognition elements within a measurement area, the abovementioned requirement can be met substantially inter alia by carefully choosing said recognition elements and the binding and detection reagents used in the assay.
In the case of arrays for assays with an inverted assay architecture, i.e. with samples of biological origin and complex composition which are immobilized in the measurement areas, meeting the above requirement is difficult and hardly reliable, since the applied samples have an unknown composition with a multiplicity of different compounds of the biological sample matrix. Therefore it is to be expected that unspecific binding of binding reagents and optionally used detection reagents can occur to a significant extent even within the measurement areas.
It is the object of the present invention to provide, for such assays with arrays of measurement areas in which samples of biological origin and complex composition which contain analytes to be detected have been immobilized, a process which enables the proportion of signals emitting from the measurement areas that are optical signals generated by unspecific interaction with the added binding reagents and with the optionally added detection reagents to be determined.
Moreover, the present invention achieves the even more general object of determining the absolute amounts of one or more analytes in immobilized samples of biological origin and complex composition and of calibrating the signals being produced due to binding reagents binding to the analytes to be detected. Generating calibration curves for signals being produced from analytes present in supplied samples binding to their recognition elements immobilized in capture arrays by means of adding a suitable number of calibration solutions which contain the corresponding analytes in suitable concentrations is well known. Disadvantageously, this method requires the addition of a multiplicity of solutions to a corresponding multiplicity of arrays of measurement areas that are very similar to one another. The international applications, WO 01/092870 and WO 02/40998, propose that in one or more arrays in each case a plurality of measurement areas with biological or biochemical or synthetic recognition elements immobilized there at a different, controlled density are provided for detection of an analyte common to said measurement areas. Particular preference is given here to the fact that, with the binding signals between an analyte and its biological or biochemical or synthetic recognition elements being known to be a function of concentration and a sufficiently large “variation” of said recognition elements immobilized at a different controlled density in various measurement areas of an array, a calibration curve for said analyte may be generated even by means of adding a single calibration solution to said array. However, these different calibration methods for capture arrays always serve to calibrate the signals due to binding of the analytes present in an unknown concentration in solution to the immobilized recognition elements of the array. In contrast, for arrays with samples of biological origin and complex composition which are immobilized in discrete measurement areas, the problem is that of calibrating the analytes present in an unknown concentration in the immobilization matrix. This problem is solved by an array according to the invention of measurement areas and a quantitative assay of the invention based thereon. By way of combination with the abovementioned process of the invention for distinguishing proportions of signals generated by specific and unspecific binding, this enables absolute amounts and concentrations of analytes to be determined in a reliable manner.
Surprisingly, the invention allows the relative and/or absolute amount or relative and/or absolute concentration of analytes present in the immobilized samples of complex composition to be determined with high accuracy also for the kind of arrays used herein.